In this renewal application entitled "influenza Immunity: Protective Mechanisms Against Pandemic Respiratory Virus," we intend to build on the progress we have made in the previous granting period with two overall objectives in mind: the first is to extend our knowledge of the human immune response to influenza vaccine much more broadly and deeply across different age groups and with different vaccine modalities and to probe the influence of genetics on these responses using monozygotic and dizygotic twin pairs. A second and new theme that we are pursuing is to use our analyses of responses to the two licensed influenza vaccines to establish a unique dataset and "metrics" of human immunology in which we can begin to identify biomarkers for what is a healthy and effective response versus what is not. In our reformulated team, we have significant strengths in immunology, infectious diseases, vaccinology, molecular biology, immune monitoring, bioengineering, genetics and bioinformatics. We also have an outstanding clinical core that has been extremely good at recruiting and processing subjects of all ages into our vaccine studies. Enhancing our clinical cohort as well as a new relationship with SRI International, which has a large and expanding twin registry in innate and adaptive immunity, the members of which will form the core of our subject base. In order to extract the maximum amount of information from each clinical specimen, we have added two new cores to our infrastructure-the Stanford Human Immune Monitoring Core to provide an in depth and uniform immunological analysis of each subject, and a Bioinformatics Core to organize the large quantities of data and to help investigators interpret it. In our projects, we focus on B and T cell responses and repertoire as well as biomarkers and signaling pathways, and in our Technology Development projects, we seek to push the boundaries of repertoire and single cell analysis and also to develop innovative new bioinformatics tools that will be vital to understanding the complexities of the immune system and the response to the influenza virus. RELEVANCE (See instructions): The Influenza virus represents a serious public health concern not just because of the illness and death that it causes but also in its potential for much greater harm in the form of a pandemic or a weapon of bioterrorism. In this renewal application, we seek to better understand how our current influenza vaccines work in terms of their interactions with the human immune system and how that interaction can be utilized to define "metrics" of immunological health. PROJECT 1: B Cell Immunity to Influenza (GREENBERG, H) PROJECT 1 DESCRIPTION (provided by applicant): A formidable and virtually unique challenge in influenza vaccine development is that the human population is repeatedly exposed to changing influenza viruses, which render previously immune individuals vulnerable to newly emerged strains, thus causing annual epidemics or even pandemics of influenza. While the human immune system has the intrinsic capability of coping with highly diversified viral antigens with its enormous antibody repertoire (estimated at a magnitude of 1011), it is our hypothesis that this potential diversity is significantly restricted in certain age groups, such as the elderly and the very young, and is not fully realized following immunization with certain types of vaccine in some age groups, such as the trivalent inactivated influenza vaccine (TIV) compared to the live attenuated influenza vaccine (LAIV) in young children^ In this application we propose to. address these, issues using, both serologic and molecular approaches, with an emphasis on the age groups most vulnerable to influenza morbidity and mortality - very young children and the elderly. The specific aims of the proposal are: A1. To compare the antibody responses against the vaccine strains (homotypic reactivity) and mismatched virus strains (heterovariant reactivity) after immunization with LAIV or TIV. We will characterize and compare the heterovariant antibody responses in a collection of paired serum samples from young children, healthy adults and elderly who are vaccinated with these two types of influenza vaccines. We will also clone and express immunoglobulin (Ig) genes from individual antibody-secreting cells (ASCs) elicited shortly after immunization with TIV or LAIV in the different age groups and compare the specificity and affinity of these monoclonal antibodies against both homotypic and heterovariant influenza viruses. A2. To identify factors affecting the sequences of Ig genes encoding influenza-specific antibodies and determine the relationship between specific Ig gene sequence usage and function, we will carry out a systematic analysis of the sequences of Ig genes isolated from ASCs after immunization with LAIV or TIV, and relate the sequence characteristics of Ig genes to antibody reactivity against different influenza strains. In particular, we will compare the Ig gene sequences between the elderly, younger adults and children, between the recipients of LAIV and TIV and between IgG and IgA isotypes. RELEVANCE (See instructions): This study is directly relevant to the design and administration of influenza vaccines for the prevention of pandemic and epidemic influenza, especially in the very young children and the elderly. It will also provide important information regarding the mechanisms responsible for the generation of protective B cell immunity against a wide array of other important human pathogens and bioterrorism agents.